Abstract Despite the critical importance of the O-glycoprotein apolipoprotein E (APOE) on Alzheimer?s disease (AD) risk, understanding of the precise mechanism behind this is limited; hence we are unable to mitigate this risk. We have been investigating APOE O-glycosylation and its changes in AD and have developed a detailed quantitative targeted mass spectrometric approach for the analysis of all APOE glycoforms at all glycosites. An understanding of glyco-APOE is essential given that glycosylation and in particular, sialic acid, has been shown to be important for its lipoprotein binding properties. We have previously shown that APOE modification is dysregulated in the healthy human APOE4.4 brain, compared to APOE3.3 individuals. We also know that transferases, the enzymes that add the monosaccharide units to glycans, wane with age and sialyltransferases have been shown to be reduced in AD. This leads us to hypothesize that APOE is glycosylated in an isoform dependent manner and that aberrant glycosylation alters A? binding, exacerbating AD pathogenesis. Our previous O-glycoproteomic analyses has shown that APOE glycosylation differs dramatically between CSF and plasma APOE, particularly in the lipid binding domain. In Aim 1 we will use human induced pluripotent stem cell (iPSC)- derived astrocytes and hepatocytes, the major sources of APOE, and characterize the glycoprofile of the APOE produced and its lipoprotein binding properties, gaining an understanding of the importance of specific glycosylation to APOE function. In Aims 2 and 3 we will focus on APOE expressed in the brain, comparing AD and healthy iPSCs and how the genetic changes we create modify APOE glycosylation and A? binding. In Aim 2 we will determine how APOE genotype affects APOE glycosylation and the A? binding properties of APOE by altering the APOE genotype of each cell line so we have each genotype with the same genetic background using the CRISPR/Cas9 system. Our preliminary glycoproteomic data also suggests that glyco-APOE is altered in AD, showing a reduction in sialylated core 1 glycans. In Aim 3, we will utilize the cell lines created in Aim 2 and modify sialyltransferase expression in healthy and AD iPSCs of all APOE genotypes. We will determine the glycosylation changes that impact A? binding and, therefore, may be involved in AD pathogenesis. In Aim 4, we will address if the cell lines produced in Aim 3 have an effect on neuronal network activity as well as amyloid accumulation by co-culturing the astrocyte cell lines with iPSC-derived neurons. Our micro-electrode array (MEA) analyses have shown that the APOE genotype of added astrocytes affects neuronal network activity making it an effective approach to test the glia impact on neurons. We will use MEAs and measures of A? accumulation to determine the effect of these aberrantly glycosylated cell lines on co-cultured neurons. Ultimately, we will elucidate the APOE glycosylation required for A? binding for each APOE isoform and how this is disrupted in AD. We will also understand the effect of aberrant APOE glycosylation on healthy and AD co-cultures and on properties relating to AD pathogenesis, determining if APOE glycosylation is primarily an AD marker or involved in AD pathogenesis.